The ULBPs (UL16 binding proteins) are a novel family of human, MHC class I-related cell-surface proteins. ULBP1 was identified as a polypeptide that bound to the human cytomegalovirus (HCMV) glycoprotein, UL16 (Cosman et al., 2001, Immunity 14:123-133). ULBP2 and ULBP3 were subsequently discovered and have some homology to ULBP1 (Id.). ULBP polypeptides share some, but not all of the features of MHC Class I proteins. The ULBPs have alpha-1 and alpha-2 domains characteristic of MUC class I proteins, but lack an alpha-3 domain and do not associate with beta-2 microglobulin. Id.
Some members of another family of human nonclassical MHC Class I proteins, the MICs, also bind UL16. Groh et al. (1996) PNAS USA 93: 12445. MICA and MICB polypeptides share some similar properties with the ULBPs, as discussed below.
The ULBPs and MICs are important activators of natural killer (NK) cells, which are a key component of the innate immune system. Activated NK cells recognize and lyse targeted cells, such as virus-infected and neoplastic cells.
NK cells recognize signals from cellular targets via receptors that are specific for MHC class I molecules on the target cell. These NK cell receptors include the killer cell Ig-like receptor (KIR), Ly49, and NKG2 receptor families. Depending on the structure of the receptor, engagement with a specific ligand will deliver either activating or inhibitory signals to the NK cell. Lanier (1998), Ann Rev Immunol 16: 359. Until recently, it was thought that signals generated by inhibitory NK cell receptors (KIRs) were dominant over those generated by any activating receptor, so that cells with downregulated MHC class I levels would be killed, according to the “missing-self” hypothesis. Ljunggren et al. (1990), Immunology Today 11: 237. However, expression of the activating ligands, ULBPs or MICs, on NK cell-resistant, MHC class I-expressing target cells renders the cells susceptible to NK cell killing. Cosman et al., supra; Bauer et al. (1999), Science 285:727-29. In addition, soluble, recombinant forms of the ULBPs, when administered to human NK cells, have now been found to bind to the NK cells and stimulate NK cytotoxicity against tumor targets. Kubin et al. (2001), Eur. J. Immunol. 31: 1428-37. ULBPs and MICs transduce an activating signal to NK cells that can override a negative signal generated by engagement of inhibitory receptors for MHC class I antigens.
ULBPs have been found to induce NK cell production of the cytokines IFN-gamma, GM-CSF, TNF-alpha, and TNF-beta, and the chemokines MIP1-alpha, MIP1-beta, and 1-309. Co-stimulation of NK cells with IL-12 has a superadditive effect on production of these factors. Cosman et al., supra; Kubin et al., supra.
MICA expression is upregulated in certain epithelial tumors, in HCMV-infected cells, and in response to stress. Groh et al. (1996), PNAS USA 93: 12445-50; Groh et al. (1999). PNAS USA 96: 6879-84. In contrast to the MICs, ULBP messages are expressed by a wide range of cells, tissues, and tumors, and on various cell lines (Cosman et al., supra). Thus, several types of cells may potentially deliver ULBP-mediated signals to NK cells and be targets of ULBP-mediated killing.
Although the amino acid sequences of the ULBPs and MICs are only distantly related, both families of proteins deliver an activating signal to NK cells by binding to NKG2D/DAP10 heterocomplexes. NKG2D is a homodimeric, C-type lectin that is expressed not only on human NK cells, but also on human CD8+ αβ T cells and γδ T cells. NKG2D expression has also been reported on murine NK cells and on activated murine CD8+ αβ T cells and macrophages. Bauer, supra; Diefenbach et al. (2000), Nature Immunol. 1: 119-126). In T cells, NKG2D acts as a costimulatory receptor, in a similar manner as CD28. Groh et al. (2001), Nature Immunol. 2: 255. The cytoplasmic domain of NKG2D is short, and signaling is mediated through its association with the DAP10 membrane adapter protein. Wu et al. (1999), Science 285:730-32. DAP10 can bind the p85 subunit of PI 3-kinase and the adapter protein Grb2. Wu et al., supra; Chang et al. (1999), J.Immunol. 163: 4651-54.
ULBP1, 2, and 3 polypeptides bind to recombinantly expressed NKG2D/DAP10 heterodimers. Anti-NKG2D antibodies block binding of ULBP 1, 2, and 3 to NK cells. Cosman et al., supra; Sutherland et al. (2002), J. Immunol. 168(2): 671-79. This evidence supports a conclusion that NKG2D is the receptor expressed on primary human NK cells that recognizes ULBP.
Agents that are effective to activate NK cell, T cell, or macrophage activity, to induce cellular production of chemokines and cytokines, and to induce target cell cytotoxicity are useful for target cell lysis, particularly for lysis of pathogen-infected cells and tumor cells. New ligands having the ability to activate NK cells, T cells, macrophages, particularly via the NKG2D/DAP10 receptor complex, are useful as agents for activating therapeutic responses from immune effector cells, for example by eliciting NK cell and/or T cell killing and other NK cell and/or T cell dependent therapies. NKG2D/DAP10 receptors are expressed on γδ T cells, CD8+ T cells, and macrophages. Bauer et al (1999), Science 285: 727-29; Diefenbach et al. (2000), Nature Immunology 1(2): 119-26. Engagement of these receptors can stimulate T cell proliferation, cytotoxicity, and cytokine production. Groh et al. (2001), Nature Immunol. 2:255; Das et al. (2001), Immunity 15:83-93.